Diabetes is a well-established risk factor for cardiovascular disease mortality which contributes to >700,000 deaths in the U.S. annually. Due to the considerable economic burden of diabetes, and the sizeable contribution of diabetes to cardiovascular disease mortality, understanding the mechanisms involved in insulin resistance is of considerable importance. Intramuscular triglyceride (IMTG) concentration may be an important mediator of skeletal muscle insulin resistance as the content of IMTG is negatively associated with insulin stimulated glucose disposal in most all populations. However, a dichotomy exists in the relationship between IMTG content and insulin sensitivity in endurance athletes and the rest of the population, as athletes have similar IMTG content as in Type II diabetics, yet are very insulin sensitive. This makes studying endurance athletes a useful tool to unraveling the relationship between skeletal muscle IMTG and insulin action. We aim to study the relationship between intramuscular triglyceride (IMTG) turnover, intracellular signals decreasing insulin action, and insulin sensitivity in endurance trained athletes, untrained lean controls, and Type II diabetics. We hypothesize IMTG turnover is decreased in Type II diabetes, leading to increased formation of long chain acyl-CoA (LCA-CoA), ceramide, and diacylglycerol (DAG). These intracellular signals could then attenuate insulin action by decreasing Akt activity and increasing PKC epsilon and theta activity which all act to decrease insulin signaling. Our first specific aim investigates intramuscular triglyceride (IMTG) turnover, intracellular signals decreasing insulin action, and insulin sensitivity during rest, exercise at 50% VO2max, and recovery. We hypothesize increased IMTG turnover in athletes compared to Type II diabetics and controls protects against decreased insulin action commonly observed with increased IMTG stores by decreasing concentration of LCA-CoA, DAG, and ceramide. The second specific aim involves an acute increase in IMTG content at rest via intralipid/heparin infusion during a hyperinsulinemic/euglycemic clamp. We hypothesize IMTG content will increase similarly in athletes and type II diabetics, while insulin action will decrease more dramatically in endurance athletes. The more dramatic decrease in insulin action will be related to a greater increase in LCA-CoA, DAG, ceramide, and Munc18c content. The significance of these studies will be to further the understanding of mechanisms promoting insulin resistance to advance the treatment and prevention of Type II diabetes.